Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation.

Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8024, USA.

Abstract

The insecticidal nature of Cry delta-endotoxins produced by Bacillus thuringiensis is generally believed to be caused by their ability to form lytic pores in the midgut cell membrane of susceptible insect larvae. Here we have analyzed membrane-associated structures of the 65-kDa dipteran-active Cry4Ba toxin by electron crystallography. The membrane-associated toxin complex was crystallized in the presence of DMPC via detergent dialysis. Depending upon the charge of the adsorbed surface, 2D crystals of the oligomeric toxin complex have been captured in two distinct conformations. The projection maps of those crystals have been generated at 17A resolution. Both complexes appeared to be trimeric; as in one crystal form, its projection structure revealed a symmetrical pinwheel-like shape with virtually no depression in the middle of the complex. The other form revealed a propeller-like conformation displaying an obvious hole in the center region, presumably representing the toxin-induced pore. These crystallographic data thus demonstrate for the first time that the 65-kDa activated Cry4Ba toxin in association with lipid membranes could exist in at least two different trimeric conformations, conceivably implying the closed and open states of the pore.

Two types of 2D crystals imaged in negative stain. (A) The 65-kDa Cry4Ba toxin crystal grown in DMPC on the air glow discharged carbon surface, and (B) the toxin crystal in DMPC attached on the hydrophobic carbon surface. Crystals were up to 1×1 μm in size. Insets show Fourier transforms of these crystalline areas.

Projection maps of propeller and pinwheel crystal forms. The maps belong to p3 plane group. The contours of the propeller-like and pinwheel-like crystals were plotted at 20 Å (A) and (B), and at 15 Å resolution (C) and (D). Insets show projection density maps of the propeller-like (C) and pinwheel-like structures (D).